Polypeptides and methods for making the same

ABSTRACT

An isolated protein having at least 90% homology with the dimeric protein having the following amino acid sequence (I) with the proviso that at the C-terminal end of the monomeric forms, 2 to 10 amino acid distance from Z 2 , a Cys is present for forming the dimeric or multimeric protein, Z 1  is a signal peptide for protein transport to the ER and/or through the plasma membrane or NH 2  or a derivative of an NH 2  group such as an alkylated, or acylated derivative, or polyethylene glycol (PEG) moiety, Z 2  is an amino acid residue with up to 20, in particular 10 or 8 amino acids which are selected of substantially non-hydrophobic naturally occurring amino acids or COOZ 3  wherein Z 3  is hydrogen, a metal ion, a hydrocarbon moiety substituted or non-substituted with heteroatoms.

This is a 371 of PCT/EP2004/001986 filed 27 Feb. 2004.

The present invention relates to soluble proteins useful for controllingT cell activation, and more particularly to soluble CTLA4 proteinsproduced by recombinant DNA methods.

BACKGROUND OF THE INVENTION

Inappropriate T cell activation has been implicated in a number ofdeleterious conditions, including autoimmune diseases and transplantrejection. Optimal activation of T cells for clonal expansion isbelieved to require two signals. One is an antigen-specific signaldelivered through T cell receptors (TCR), while the second is anantigen-non-specific or co-stimulatory signal.

The second non-specific signal is determined by a class of T cellregulatory molecules known as co-stimulators that determine whether Tcells are activated or not. B7 (1 and 2), are co-stimulatory proteinsexpressed on the cell surface of antigen presenting cells such asmacrophages, B lymphocytes and dendritic cells as reported.

B7 (1 and 2) is a natural ligand for T cell surface proteins known asCD28 and CTLA4 (cytolytic T-lymphocyte-associated antigen number 4,CD152). CD28 and CTLA4 share substantial homology in their amino acidsequences, particularly in the transmembrane and cytoplasmic domains,and are therefore believed to share similar functions in the T-cellco-stimulation pathway. B7 is known to have a greater affinity for CTLA4compared with CD28.

CD28 is constitutively expressed on most T lymphocytes. CTLA4, however,was determined to be preferentially expressed by activated versusunactivated T cells.

The interactions of B7 with CD28 and CTLA4 play an important role in thefull activation of T cells in the co-stimulation pathway during animmune response. Neutralisation of B7 or CD28 activity, for example withmonoclonal antibodies, prevents T cell proliferation. Neutralisation ofB7 activity also prevents T cells from acting as helper cells for theinduction of antibody synthesis by B cells.

However, previous attempts to express the extracellular domain of CTLA4as a soluble, unfused protein have been unsuccessful. According to PCTPublication No. WO 93/00431, successful expression of active CTLA4proteins is believed to require an expression system that permits theproteins to form as dimers because the proteins are believed to occur innature as dimers. Thus, researchers have focused on fusion proteins inan effort to find an appropriate expression system.

A fusion protein containing the extracellular domain of CTLA4 joined tothe Fc heavy chain region of an immunoglobulin molecule has beendeveloped as a possible agent having B7 inhibitory activity. This fusionprotein, referred to as “CTLA4 Ig fusion protein,” is described inLinsley et al., J. Exp. Med. 174:561-569 (1991) and in PCT PatentPublication No. WO-A-93/00431.

According to these publications, the CTLA4-Ig fusion protein is secretedfrom mammalian cells as a disulfide-linked dimeric protein thataggregates in solution. However, attempts to express the extracellulardomain of CTLA4 as an unfused protein in mammalian cells wereunsuccessful. The Ig portion of the fusion protein facilitates theformation of a dimeric fusion protein, which is capable of beingprocessed and expressed by the mammalian cells. The Ig portionadditionally allows the active fusion protein to be purified fromconditioned media using a protein A affinity column. Protein A has ahigh affinity for the Fc region of Ig molecules.

The molecular weight of the major CTLA4-Ig species in solution isapproximately 200 kDa based on size exclusion chromatography alsodescribed in Linsley et al., J. Exp. Med. 174:561-569 (1991). Becausethe molecular weight of the monomeric form of the CTLA4-Ig fusionprotein expressed in mammalian cells is about 50 kDa, the major speciesin solution is believed to be an aggregated complex of at least fourCTLA4-Ig molecules.

The B7 inhibitory activity of the CTLA4-Ig fusion protein has beentested in both in vitro and in vivo experiments. In the in vitroexperiments, the CTLA4-Ig fusion protein bound to B7 and neutralised itsactivity. The CTLA4-Ig fusion protein was found to inhibit T cellproliferation. The fusion protein also inhibited the ability of helper Tcells to stimulate antibody production by B lymphocytes in an in vitrostudy described in Linsley et al., J. Exp. Med. 174:561-569 (1991).

In experiments conducted in vivo, the CTLA4-Ig fusion protein wasdetermined to be immunosuppressive and capable of prolonging survival ofpancreatic and heart allografts in mice and rats. In mouse studies, theadministration of CTLA4-Ig resulted in the long term acceptance ofallografts. These results suggest that the fusion protein had tolerizedthe recipient mice to the foreign tissue.

The CTLA4-Ig fusion protein has several disadvantages as a therapeuticagent for human disease. Because the fusion protein is a non-naturallyoccurring molecule, a patient receiving the protein may develop animmune response to the protein.

Antigenicity may be more of a problem when patients are taken off thetherapeutic agent so they are no longer immunosuppressed and are capableof mounting an immune response against the fusion protein. Therefore,antigenicity may prevent the CTLA-Ig fusion protein from beingadministered intermittently to patients suffering from chronic diseases.In addition, the half-life of the CTLA4-Ig fusion protein in mice isabout 4 days, with significant levels of the fusion protein stilldetectable in the animals 5 weeks after the cessation of treatment withCTLA4-Ig. Furthermore, when bound to B7 on the surface ofantigen-presenting cells, the Ig portion of the fusion protein mayactivate the complement cascade that results In cell death andhematological problems.

Thus, a need exists for additional therapeutic agents capable ofinhibiting the co-stimulatory pathway in T cell activation. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to recombinantly-produced CTLA4polypeptides that are not fusion proteins containing human Ig molecules.The soluble, recombinant polypeptides contain, as a basic unit, amonomer consisting essentially of the extracellular domain of the CTLA4receptor protein. Preferably, the recombinant polypeptides are theproduct of joining two or more monomers through intermolecular sulfidebonds to form biologically active dimers or multimers.

The polypeptides can also be functional derivatives of the monomers andmultimers, such as, for example, pegylated molecules.

The present invention also provides methods of making the recombinantpolypeptides. The methods include the steps of:

(a) obtaining a DNA sequence capable of directing a host cell to expressa polypeptide corresponding to the extracellular domain of a CTLA4receptor protein, the polypeptide having B7 binding activity;

(b) inserting the DNA sequence into a vector having operational elementsfor expression of the DNA sequence;

(c) transferring the vector into a host cell capable of expressing thepolypeptide;

(d) culturing the host cell under conditions for expression of thepolypeptide;

(e) harvesting the active polypeptide.

Vectors and host cells useful for the expression of the recombinantCTLA4 polypeptides are also provided. In addition, the invention furtherprovides pharmaceutical compositions containing the CTLA4 polypeptidesas the active ingredient.

The invention is related with an isolated protein having at least 90%homology with the dimeric or multimeric protein having the followingamino acid sequence wherein the dimer has the following structures:

with the proviso that at the C-terminal end of the monomeric forms, 2 to10 amino acid distance from Z², a Cys is present for forming the dimericor multimeric protein.

Z¹ is a signal peptide for protein transport to the ER and/or throughthe plasma membrane or NH₂ or a derivative of an NH₂ group such as analkylated, or acylated derivative, or polyethylene glycol (PEG) moiety,

Z² is an amino acid residue with up to 20, in particular 10 or 8 aminoacids which are selected of substantially non-hydrophobic naturallyoccurring amino acids or COOZ³ wherein Z³ is hydrogen, a metal ion, ahydrocarbon moiety substituted or non-substituted with heteroatoms. Themonomeric forms may be linked by Cys-Cys bonds or e.g. crosslinked PEGmoieties to form di- or multimeric forms. The protein of the inventionmay be altered by exchange of amino acids as long as the protein isstill functional. In most cases this is the case when 90% homology ismaintained. The modification of the amino acid chain may be achieved byreplacing single amino acids or clusters of amino acids of the sequenceor deletions. An exchange of amino acid can in one way be done by aconservative exchange of for example an aromatic amino acid againstanother aromatic amino acid or by exchange of a cationic amino adds. TheZ² substituent should not be too non-polar in order to avoid alipophilic area which may cause binding of the protein in membranes.

Subject matter of the invention is also the monomeric part of thedimeric protein of the invention having the following amino acidsequence:

(SEQ ID NO: 1) Z¹mhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymtgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpds-Z²wherein Z¹ and Z² have the same meaning as mentioned above.

The monomer is important since this is coded by a nucleic acid in thecell. The expression of the respective gene leads after posttranslational modification by the cell to the protein of the invention.Multimeric forms can be formed, starting from this monomer.

A particular signal peptide of the protein of the invention comprises aZ¹ residue of the following sequence:

(SEQ ID NO: 2) maclgfqrhk aqlnlaartw pctllffllf ipvfcka.

In a particular embodiment of the invention the protein of the inventionis soluble under physiological conditions.

In another embodiment the protein of the invention wherein comprises anextra cellular domain of the CTLA4 protein.

The protein of the invention can bind B7-1 or B7-2.

An isolated nucleic acid coding for the monomeric protein of theinvention is also subject matter of the invention. The expression of therespective gene leads after post translational modification by the cellto the protein of the invention.

The expression of the gene in a bacterial or eukaryotic host cell leadsto the monomeric protein which dimerises in the cellular medium. Whensecreted the signal peptide is removed. The nucleic acid may contain anucleotide sequence encoding a CTLA4 extracellular domain.

A recombinant expression vector comprising a nucleic acid is alsosubject of the invention. The vector can be used for transfection of ahost cell directing the expression of the protein of the invention ordirecting the expression of a CTLA4 extracellular domain.

The protein of the invention can be formulated in a composition suitablefor pharmaceutical administration. The pharmaceutical formulationcomprises a protein of the invention, and a pharmaceutically acceptablecarrier.

According to the invention the protein of the invention can be producedby a method comprising culturing a host cell of the invention in amedium to express the protein and isolating the protein from the mediumor purifying the protein from inclusion bodies.

In another embodiment the inventions concerns a method for producing afusion protein consisting of a protein of the invention with animmunoglobulin protein comprising culturing a host cell of the inventionin a medium to express the protein and purifying the protein by releasefrom periplasm.

The invention claims also a method for inhibiting an interaction of aCTLA4 ligand on an antigen presenting cell with a receptor for the CTLA4ligand on a T cell the method comprising contacting the antigenpresenting cell with a protein of the invention, a method for treatingan autoimmune disease in a subject mediated by interaction of a CTLA4ligand on an antigen presenting cell with a receptor for the CTLA4ligand on a T cell, comprising administering to the subject the proteinof the invention.

In particular the invention is concerned with a method, wherein theautoimmune disease is selected from the group consisting of diabetesmellitus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis,systemic lupus erythematosis, psoriasis vulgaris, myasthenia gravis,Graves' disease, Goodpastures' disease, idiopathic thrombocytopeniapurpura (ITP), aplastic anemia, inflammatory bile disease, idiopathicdilated cardiomyopathy (IDM) and autoimmune thyroiditis.

In a further embodiment of the invention the method for treating allergyin a subject is mediated by interaction of a CTLA4 ligand on an antigenpresenting cell with a receptor for the CTLA4 ligand on a T cell,comprising administering the protein of the invention to the subject.

In another embodiment a method is disclosed for inhibitinggraft-versus-host disease (GVHD) in a bone marrow transplant recipient,comprising administering to the recipient the protein of the invention.

In yet another embodiment a method is disclosed, wherein donor bonemarrow is contacted with the protein of any one of the claims 1 to 6 andwith cells from the transplant recipient ex vivo prior totransplantation of the donor bone marrow into the recipient.

In still another embodiment antigen presenting cells of the recipient iscontacted with the protein of any one of the claims 1 to 6 and withcells, most preferably T cells from the donor.

Another embodiment is concerned with a method for inhibiting rejectionof transplanted cells in a transplant recipient, comprisingadministering to the recipient the protein of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides soluble proteins of the CTLA4 type, e.g.recombinantly produced, that are not fused to human Ig molecules proteinof the present invention is useful for inhibiting inappropriate T cellactivation that can lead to various disorders.

The naturally-occurring or wild-type CTLA4 protein is a ligand of B7,which is a cell surface protein involved in the costimulatory pathwayleading to T cell activation. The nucleotide and amino acid sequences ofmurine and human CTLA4 are reported Brunet, J. F., Denizot, F., Luciani,M. F., Roux-Dosseto, M., Suzan, M., et al. 1987. A new member of theimmunoglobulin superfamily—CTLA-4. Nature 328:267-70. Dariavach, P.,Mattei, M. G., Golstein, P., Lefranc, M. P. 1988. Human Ig superfamilyCTLA-4 gene: chromosomal localization and identity of protein sequencebetween murine and human CTLA-4 cytoplasmic domains. Eur J Immunol18:1901-5. The overall amino acid homology between human and murineCTLA4 proteins is 76%. The correct amino acid sequence of the fulllength human CTLA4 protein Is provided in PCT Publication No.WO-A-93/00431.

As noted previously, earlier attempts to produce an unfused or truncatedCTLA4 protein have been unsuccessful. Therefore, prior to the presentinvention, methods for obtaining a biologically active, recombinantCTLA4 protein involved expressing CTLA4 as a fusion protein. Moreparticularly, the fusion protein is described in PCT Publication No.WO-A-93/00431 as containing the extracellular domain of CTLA4 fused tothe heavy chain region of a human immunoglobulin molecule (referred toas “CTLA4-Ig” protein). According to this publication, successfulexpression of the extracellular domain of the CTLA4 receptor proteinrequires an expression system that permits the protein to form dimers.In contrast, the unfused or truncated versions of the CTLA4 proteinappeared not to be expressed in an active form in eukaryotes. Incontrast, the protein of the invention, however, is an active truncatedversion of the CTLA4 protein can be produced in eukaryotes. Thepublication further indicates that the Ig portion of the CTLA4-Ig fusionprotein is believed to facilitate dimer formation and to aid in thepurification of the fusion protein by conventional protein A affinitychromatography.

The present invention is based on methods for producing biologicallyactive, soluble proteins of the CTLA4-type (sCTLA4) that are not fusedto an Ig chain. As used herein, the term “biologically active” refers topolypeptides that exhibit B7 binding activity.

The protein of the invention has, as a basic unit, a monomer thatconsists essentially of the extracellular domain of the wild-type CTLA4receptor protein, preferably including the cysteine close to thetransmembrane domain. In reference to monomers, the term “consistsessentially of” as used herein is intended to encompass a monomerencoded by an amino acid sequence corresponding to the extracellulardomain of the wild-type CTLA4 protein or corresponding to theextracellular domain joined to additional amino acids other than anamino acid sequence encoding for a complete human Ig molecule.

The protein of the present invention can also be in the form of dimersor other multimers, which contain more than one basic monomeric unit.Such multimers, particularly dimers, can be formed by joining two ormore monomers through intermolecular disulfide bonds or by crosslinkingagents such as, for example, polyethylene glycol (hereinafter referredto as “PEG”), other polyethers, EDTA and other linkers known to thoseskilled in the art.

The present invention further provides methods of producing the proteinof the present invention. Such methods include the steps of:

(a) obtaining a DNA sequence capable of directing a host cell to expressa polypeptide corresponding to the extracellular domain of a CTLA4receptor protein, the polypeptide having B7 binding activity;

(b) inserting the DNA sequence into a vector having operational elementsfor expression of the DNA sequence;

(c) transferring the vector into a host cell capable of expressing thepolypeptide;

(d) culturing the host cell under conditions for expression of thepolypeptide.

Optionally, the peptide can thereafter be permitted to assume aquaternary structure in which two or more monomers join to form a unit,such as a dimer or other multimeric forms. In addition, the presentinvention further optionally includes separating the dimeric forms toobtain the form with the most inhibitory activity, referred to herein asthe active dimeric form.

As used herein, the term “functional equivalent(s)” means modifiedsequences having one or more additions, deletions, or substitutions tothe above sequence that do not substantially affect the ability of thesequence to encode a polypeptide having B7 binding activity. Suchmodified sequences can be produced by means known in the art, including,for example, site directed mutagenesis. The sequences can be obtainedfrom natural sources, such as the natural DNA sequence encoding theextracellular domain of a CTLA4 receptor protein. Alternatively, thesequence can be produced synthetically according to methods known in theart. Additionally, such DNA sequences can be derived from a combinationof synthetic and natural sources. The natural sequences further includecDNA and genomic DNA segments. Methods of obtaining the synthetic andnatural DNA sequences are described in PCT Publication No.WO-A-93/00431, which is incorporated herein by reference.

Vectors that can be used in these methods include those vectors intowhich a CTLA4 DNA sequence has been introduced, as described above. Asused herein, the term “consisting essentially of” In reference tovectors means that such vectors contain nucleotide sequences that encodefor the extracellular domain of CTLA4 receptor protein, including anydesired operational elements, but not nucleotide sequences encoding ahuman Ig molecule. A CTLA4 DNA sequence can be inserted and linked withany desired operational elements to effect its expression. The vectorscan contain one or more of the following operational elements: (1) apromoter; (2) a Shine Dalgarno sequence and initiator codon; (3) aterminator codon; (4) an operator; (5) a leader sequence to facilitatetransportation out of the host cell; (6) a gene for a regulator protein;and (7) any other DNA sequences necessary or preferred for appropriatetranscription and subsequent translation of the vectors.

The vectors can be transferred into suitable host cells by variousmethods known in the art, including transfection and transformationprocedures. Various transfer methods are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2000),which is incorporated herein by reference. Such host cells can be eithereucaryotic or procaryotic cells. Examples of such host cells includechinese hamster ovary (CHO) cells, Vero cells, HEK293 cells, yeast, E.coli and baculovirus infected insect cells.

The host cells of the present invention can be cultured under conditionsappropriate for the expression of the protein of the invention. Theseconditions are generally specific for the host cell and are readilydetermined by one of ordinary skill in the art In light of the publishedliterature regarding the growth conditions for such cells.

In one embodiment, cells can be grown to a high density in the presenceof appropriate regulatory conditions that inhibit expression of thedesired protein of the invention. When optimal cell density is reached,the environmental conditions can be altered to those appropriate forexpression of the polypeptide according to procedures known in the artor as described in the examples below. Therefore, prior to harvestingthe expressed protein of the invention, it is particularly useful toallow the host cells to grow near optimal density before inducingexpression.

Expression of the protein of the invention can be confirmed by usinganti-CTLA4 antibodies according to assay procedures known in the art,such as Western blotting or ELISA for example. Once expression of therecombinant polypeptides has been confirmed, the polypeptides can thenbe harvested according to methods known to those skilled in the art.

The recombinant polypeptides can be purified after harvesting.

The present invention also provides functional derivatives of theprotein of the invention. As used herein, the term “functionalderivative” means any biologically active modified form of the proteinof the invention. Such modifications can be (1) substitutions oradditions in the amino acid sequence, and/or (2) the addition of anotherfunctional group to improve certain pharmacokinetic or immunologicproperties. Such modifications, however, should not substantiallydecrease the biological activity of the parent recombinant polypeptideby no more than a 10-fold decrease, preferably less than a 5-folddecrease in activity. Therefore, as used herein, the term “functionalderivative” can mean an active fragment, an analog or a derivative ofthe protein of the invention described above that substantially retainsthe biological activity of the unmodified protein of the invention. Inthe case of analogs, such modified polypeptides preferable have an aminoacid homology of greater than about 40% compared to the extracellulardomain of CTLA4, more preferably in excess of 50%, and most preferablyin excess of 90%. An amino acid homology of about 99% is particularlyuseful.

For example, one modification can be the substitution or addition of acysteine to provide a “free cysteine” within the amino acid sequence toproduce a “cysteine mutein.” The terms “cysteine mutein” or “CTLA4mutein,” as used herein, refers to muteins having at least one cysteinethat is not involved in an intramolecular or intermolecular disulfidebond. The free cysteine can appear at any amino acid residue that doesnot substantially interfere with its ability to bind B7.

The muteins and other derivatives can be prepared by methods well knownto those skilled in the art. Such methods include, for example,mutagenic techniques in which nucleotides are substituted or added thatencode for a cysteine. Alternatively, the muteins can be synthesized bymethods also known to those skilled in the art.

In one embodiment, the cysteine mutein can be attached to polyethyleneglycol (PEG) at a free cysteine to increase its molecular weight andimprove its pharmacokinetic properties such as an increased serumhalf-life. Long chain polymer units of PEG can be bonded to the muteinvia covalent attachment to the sulfhydryl group of a free cysteineresidue on the mutein.

Various PEG polymers with different molecular weights can be used, forexample, 5.0 kDa (PEGs), 8.5 kDa (PEGs), 10 kDa (PEGs) and 20 kDa(PEGs). To obtain selectivity of reaction and homogenous reactionmixture, it is useful to use functionalized polymer units that willreact specifically with the sulfhydryl groups. The functional orreactive group attached to the long chain PEG polymer is the activatinggroup to which the mutein attaches at the free cysteine site. Suitableactivating groups include, for example, maleimide, sulfhydryl, thiol,triflate, tresylate, aziridine, exirane or 5-pyridyl. PEG molecules canalso be attached to the protein of the invention at free amines usingNHS(N-hydroxysuccinimide)-derivatized PEG molecules.

Other conjugates of the protein of the invention are also contemplated,for example, (1) by attaching a single PEG molecule to a monomer(monopegylated) or dimer, for example, at free amines; (2) by attachingtwo PEG molecules to a dimer of the protein of the invention. Thoseskilled in the art can readily determine the appropriate pH,concentration of polypeptide, and ratio of polypeptide to PEG necessaryto produce a useful yield of the pegylated polypeptide.

The present invention further provides pharmaceutical compositionscontaining the protein of the invention or its functional derivatives ina pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” as used herein means anon-toxic, generally inert vehicle for the active ingredient, which doesnot adversely affect the ingredient or the patient to whom thecomposition is administered. Suitable vehicles or carriers can be foundin standard pharmaceutical texts, for example, in Reminston'sPharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.(1980), incorporated herein by reference. Such carriers include, forexample, aqueous solutions such as bicarbonate buffers, phosphatebuffers, Ringer's solution and physiological saline. In addition, thecarrier can contain other pharmaceutically-acceptable excipients formodifying or maintaining the pH, osmolarity, viscosity, clarity, colour,sterility, stability, rate of dissolution, or odour of the formulation.

The pharmaceutical compositions can be prepared by methods known in theart, including, by way of an example, the simple mixing of reagents.Those skilled in the art will know that the choice of the pharmaceuticalcarrier and the appropriate preparation of the composition depend on theintended use and mode of administration.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form that requiresreconstitution prior to administration.

Generally, storage of the formulations is at temperatures conventionalfor such pharmaceuticals, including room temperature or preferably 40°C. or lower, such as −70° C. The formulations can be stored andadministered between a pH range of about 5 to 8, preferably at aboutphysiological pH.

The recombinant polypeptides and functional derivatives thereof can beused to prevent, suppress or treat disorders associated withInappropriate T cell activation and proliferation. Accordingly, thepresent Invention provides methods for the therapy of disordersassociated with such deleterious T cell activation and proliferation.Such disorders include, for example, transplantation rejection, variousautoimmune diseases and allergies and other T-cell mediated disorders.The autoimmune diseases for which the administration of the protein ofthe invention and functional derivatives may be useful includerheumatoid arthritis, asthma, lupus, multiple sclerosis, psoriasis,graft versus host disease, Type I diabetes and other autoimmunediseases.

The therapeutic methods of the present invention are accomplished byadministering to a patient an effective amount of a protein of theinvention or a functional derivative thereof to inhibit deleterious Tcell activation. The active ingredient is preferably formulated into apharmaceutical composition as previously described.

As used herein, the term “patient” refers to any human having T cellsthat are capable of being co-stimulated by B7. In addition, the proteinof the invention and its functional derivative is also referred to asthe “active ingredient(s).” An effective dosage depends on a variety offactors known to those skilled in the art, including the species, age,weight, and medical condition of the patient, as well as the type ofdisorder to be prevented, suppressed or treated, the severity of thecondition, the route of administration and the active ingredient used. Askilled physician or veterinarian can readily determine and prescribe aneffective amount of the active ingredient.

Generally, treatment is initiated with small dosages substantially lessthan the optimum dose of the active ingredient. Thereafter, the dosageis increased by small increments until the optimum or desired effect isattained without causing significant harm or deleterious side effects.

Preferably, the daily dosage is in the range of about 10-5000 mg perhuman patient.

The compounds and pharmaceutical compositions of the present inventioncan be administered orally or parenterally by any means known in theart, including, for example, by intravenous, subcutaneous,intraarticular or intramuscular injection or infusion. To achieve andmaintain the desired effective dose, repeated administration may bedesirable. The frequency of dosing will depend on several factors suchas, for example, the formulation used, the type of disorder, theindividual characteristics of the patient, and the like. Those skilledin the art can readily determine the appropriate frequency based on suchfactors.

The protein of the invention can also be used to contact a donor bonemarrow with the protein of the invention and with cells from thetransplant recipient ex vivo prior to transplantation of the donor bonemarrow into the recipient. More specifically, antigen presenting cellsof the recipient are incubated with the protein of the invention and arecontacted with cells, most preferably T cells of the donor. AlloreactiveT cells will be anergized, driven into apoptosis or educated to Tregulatory cells this way.

EXAMPLES

The following examples are intended to illustrate but not limit thepresent invention.

Reduction of CD86 by hCtla4 in Raji Cells

FIG. 1: Raji cells (human B cell lymphoma) were infected with a vectorcoding for hCTLA4Ig or hCTLA4Cys (sCTLA4) (proteins of the invention).Surface expression of CD86 was analysed several times. This figure showsday 7 post infection. The transfection rate was not evaluated. It canclearly be seen, that the number of CD86 low Raji cells increased, whenany of the CTLA4 constructs was expressed in the cell population. ThesCTLA4 (Ctla4Cys) was more efficient (6,3%) then the Ctla4Ig (3,6%).

Reduction of CD86 by sCtla4 in A20 Cells

FIG. 2: Transfection of A20 cells (mouse B cell lymphoma) with sCTLA4protein of the invention (Ctla4Cys) drastically reduces the surfaceamount of CD86. Increasing the sCTLA4 amount by a second transfection,reduces the CD86 amount even further. It is known that only the CTLA4dimer efficiently binds CD86.

Reduction of CD86 by the Protein of the Invention in Primary DendriticCells

FIG. 3: Primary mDC were infected at day 3 after isolation with a MOI of1 or 0.2 with virus coding for sCTLA4 (mCTLA4Cys). CD86 expression ofthese cells was measured frequently. The upper row shows mDC three daysafter infection (day 6 of culture). The lower row shows mDC eight daysafter infection. On day 7, 16 hours before the analysis, the cells weresupplemented with 0.1 ng LPS/ml. The increase of the CD86 amount in thetransfected cells was hampered by expression of sCTLA4 (Ctla4Cys).

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It should be understood that variousmodifications can be made without departing from the spirit and scope ofthe invention.

Accordingly, the following claims are intended to be interpreted, toembrace all such modifications.

1. An isolated protein of amino acid sequence

wherein Z¹ is (i) a signal peptide for protein transport to the ERand/or through the plasma membrane, (ii) NH₂, (iii) an alkylatedderivative of NH₂, (iv) an acylated derivative of NH₂, or (v) apolyethylene glycol (PEG) moiety and Z² is (i) an amino acid chainwherein the chain has up to 20 non-hydrophobic naturally occurring aminoacids, or (ii) COOZ³ wherein Z³ is hydrogen or a metal ion.
 2. Theprotein of claim 1 wherein the chain has 8 or 10 amino acids.
 3. Amonomeric protein (SEQ ID NO: 1)Z¹-mhvaqpavvlassrgiasfvceyaspgkatevrvtvlrqadsqvtevcaatymtgneltflddsictgtssgnqvnltiqglramdtglyickvelmypppyylgigngtqiyvidpepcpds-Z²,

of dimeric protein

wherein Z¹ is (i) a signal peptide for protein transport to the ERand/or through the plasma membrane, (ii) NH₂, (iii) an alkylatedderivative of NH₂, (iv) an acylated derivative of NH₂, or (v) apolyethylene glycol (PEG) moiety and Z² is (i) an amino acid chainwherein the chain has up to 20 non-hydrophobic naturally occurring aminoacids, or (ii) COOZ³ wherein Z³ is hydrogen or a metal ion.
 4. Theprotein of claim 1 wherein Z¹ is (SEQ ID NO: 2) maclgfqrhk aqlnlaartwpctllffllf ipvfcka.


5. The protein of claim 1 which is soluble under physiologicalconditions.
 6. The protein of claim 1 comprising an extracellular domainof the CTLA4 protein.
 7. The protein of claim 1 binding B7-1 or B7-2. 8.The protein of claim 3 crosslinked to PEG-moieties.
 9. A compositionsuitable for pharmaceutical administration comprising the protein ofclaim 1 and a pharmaceutically acceptable carrier.
 10. A compositionsuitable for pharmaceutical administration comprising the monomericprotein of claim 1 and a pharmaceutically acceptable carrier.